ABSTRACT Chemically modified nucleic acids (CNAs) function as potential antigene-, antisense-, or RNA interference (RNAi)-based drugs, as model systems for native DNA and RNA, as chemical probes in diagnostics and in high-throughput genomics and drug target validation, or the analysis of protein-nucleic acid interactions, and as tools for structure determination. This application is a continuation of our research directed at CNAs, with the long-term objectives to optimize their structure and activity for future applications as antisense oligonucleotide (AON) and small interfering RNA (siRNA) therapeutics, to devise an etiology of nucleic acid structure, and to determine the origins of substrate recognition by selected DNA- and RNA-processing enzymes. We propose four aims of broad biological significance in understanding the consequences of chemical modification for nucleic acid structure and stability and, by probing nucleic acid-protein interactions structurally and functionally by way of CNAs, the principles affording substrate recognition and processing by RNase H and A- and Y-class DNA polymerases. Aim (1) focuses on investigations of the conformational features underlying the stability and efficacy of modifications assessed in connection with the discovery and development of the next generation of AON and siRNA therapeutics. This work will be carried out in collaboration with two world leaders in R&D of nucleic acid drugs, Alnylam Pharmaceuticals Inc. and Isis Pharmaceuticals, Inc. In Aim (2) we will scrutinize the pairing and structure of glycol nucleic acid (GNA), the simplest artificial pairing system with a phosphate backbone found to cross-pair with RNA. We will also use neutron macromolecular crystallography (NMC) to delve deeper into aspects of nucleic acid structure that have eluded characterization using standard techniques, such as the orientations of water molecules and ribose 22-hydroxyl groups. Work in Aim (3) is directed at RNase H, an endonuclease that plays a key role in antisense applications by way of destroying the mRNA targeted by certain AONs. By way of 3D structural data for complexes with duplexes that are bound but not cleaved, we will probe features of nucleic acids central to recognition. The conformational range of the strand opposite RNA tolerated by the enzyme will be gauged with 3D structures of complexes with AON/RNA hybrids. In Aim (4) we will address the recent hypothesis that certain DNA polymerases appear to rely more on shape than hydrogen bonding for accurate and efficient replication. Building on our recent structures of CNAs containing 2,4-difluorotoluene (F, an apolar T mimic) and complexes of F-modified templates with a trans-lesion (Y-class) DNA Pol, we will determine structures of ternary Pol-DNA-dNTP complexes containing F or dFTP of a replicative (A-class) DNA Pol, and correlate these data with activity data in the pre-steady- and steady-states. The main tool to be used is X-ray crystallography. Other approaches we will rely on to achieve our objectives are synthetic organic chemistry, biochemical and molecular biology tools as well as thermodynamics, kinetics and single-crystal NMC.